Human cytomegalovirus (HCMV), like other viruses, exploits host cell machineries and, in doing so, provides powerful tools for gaining insight into cellular regulatory mechanisms. HCMV is the leading viral cause of congenital disorders in developed countries and a significant contributor of morbidity and mortality in immunosuppressed patients. Understanding the action of HCMV UL37 proteins is of high impact as they partially underlie HCMV pathogenesis. Following synthesis, UL37 proteins are translocated into the endoplasmic reticulum (ER) membrane and then imported into mitochondria, where they have potent anti- apoptotic functions. Trafficking of UL37 proteins is central to the ordered events in the viral lytic cycle, host cell survival during infection, and the assembly of infectious progeny virus. Contacts between the ER and mitochondria occur through mitochondria associated membranes (MAM) and these contacts regulate critical aspects of cell physiology. Few viral or cellular proteins are known to traffic into the MAM and, fewer yet, from the MAM to mitochondria. We have recently shown that HCMV UL37 proteins target the MAM. We hypothesize that HCMV infection exploits this pathway and, by the action of UL37 proteins, increases cellular ER- mitochondrial contacts. In Aim 1, we will determine whether and how HCMV infection, in part by UL37 exon 1 protein (pUL37x1) functions, increases ER-mitochondrial contacts. To that end, we will examine if pUL37x1- mediated calcium release or restructuring of F-actin or microtubule cytoskeletons increases ER-mitochondrial contacts and if HCMV infection increases ER-mitochondrial stabilizers. In Aim 2, we will determine how HCMV infection alters the understudied MAM composition and, thereby, identify specific, HCMV-targeted cellular proteins that enhance ER-mitochondrial contacts. For these studies, we will perform comprehensive, comparative proteomic analyses on HCMV-infected versus uninfected human diploid fibroblasts and determine if HCMV infection affects trafficking of sigma 1 receptor, a MAM constituent that responds to ER stress. We will also test whether pUL37x1 plays a role in these alterations by comparing cells infected with HCMV UL37x1 mutant or parental virus. By identifying how HCMV infection affects ER-mitochondrial contacts, we should define a novel mechanism of how HCMV enhances cell survival. Proteomic profiling should identify specific MAM protein components whose expression or location is altered by HCMV infection and, importantly, which may play critical roles in increasing ER-mitochondrial contacts, not only during infection but in uninfected cells as well. Thus, HCMV may help us identify key cellular MAM constituents and elucidate how ER-mitochondrial contacts are regulated. Further, this exploratory study should set the stage for more detailed analyses of the mode of action of UL37 proteins and the possibility that they may serve as targets for antiviral drugs. PUBLIC HEALTH RELEVANCE: Human cytomegalovirus (HCMV), a human beta-herpesvirus, is the leading viral cause of congenital infections in developed countries and a significant cause of life-threatening diseases of immunosuppressed patients. In order to grow, HCMV, like other viruses, exploits host cell machineries and, in doing so, provides powerful tools for gaining insight into cellular regulatory mechanisms. Trafficking of its UL37 immediate early proteins from the endoplasmic reticulum (ER) to mitochondria is unconventional but central to its replication and to cell survival during infection. These studies will determine how HCMV infection and, particularly, its UL37 proteins affect contacts between the ER and mitochondria, which regulate critical aspects of cellular physiology. By identifying how HCMV infection affects ER-mitochondrial contacts, we should define a novel mechanism of how HCMV enhances its growth. Understanding the action of HCMV UL37 proteins is particularly important as they partially underlie its pathogenesis. This exploratory study should set the stage for more detailed analyses of the mode of action of HCMV UL37 proteins and the possibility that they may serve as targets for intervention.